(PQC2) Niche-responsive RNA editing by ADAR1 in dormant multiple myeloma initiating cell maintenance

 描述(申请人提供)：尽管有新的治疗方法，包括免疫调节药物，如沙利度胺和来那度胺治疗多发性骨髓瘤(MM)，但约20%-25%的患者仍被认为存在治疗失败的高风险。即使在强化治疗方案下，治疗顽固性病例和疾病复发的出现也表明，骨髓(BM)中存在能够逃脱药物的潜伏的骨髓瘤始发群体。这些骨髓瘤起始细胞缺乏浆细胞标志物CD138，但它们表达多种造血干细胞(CD38)和B细胞标志物，如CD27和CD19；然而，调控骨髓瘤起始细胞生成和维持的分子和细胞机制迄今知之甚少。最近，我们和其他研究小组发现，Notch信号和炎症反应ADAR1激活都是调节骨髓微环境中恶性干细胞维持的关键事件，其特征是促进休眠前体细胞的存活和自我更新以及细胞周期的改变。在此背景下，这一点的中心假设 建议是MM小生境衍生的促炎信号通过腺苷脱氨酶作用于dsRNA-1(ADAR1)在休眠的骨髓瘤启动细胞中诱导异常的人类特异性RNA编辑，该细胞因对来那度胺的耐药性而加剧。该项目将：1)确定ADAR1活性是否在骨髓瘤启动细胞中得到增强，并研究来那度胺治疗对来那度胺依赖的RNA编辑的影响；2)确定在骨髓瘤启动细胞中激活ADAR1依赖的RNA编辑的缺口调节的促炎细胞因子；以及3)确定直接抑制MM启动细胞中的ADAR1活性，或阻断激活ADAR1的微环境信号，是否使骨髓瘤启动细胞对来那度胺敏感，并防止骨髓瘤启动细胞的维持。这些目标将解决PQC2：什么分子或细胞事件在治疗后建立肿瘤休眠，什么导致复发？我们将利用体外和体内测量骨髓瘤启动细胞功能的多色流式细胞术和基于荧光RNA探针的策略来纯化和描述主要的MM细胞成分，并结合基于qPCR的新的诊断分析来检测内源RNA编辑，以及基因敲除(CRISPR)或慢病毒shRNA敲除策略来调节缺口依赖的ADAR1激活。一种荧光泛素化细胞周期指示剂(FUCCI)双顺反子慢病毒报告系统将有助于在稳健的骨髓基质共培养模型和生物发光的人源化MM小鼠模型中研究休眠的活细胞。最终目标是研究依赖于小生境的ADAR1激活作为一种新的机制来驱动休眠的骨髓瘤启动细胞的转录组重新编码和分子进化，为靶向治疗在一系列其他治疗顽固性恶性肿瘤中的潜在应用奠定基础。

 DESCRIPTION (provided by applicant): Despite new available therapies, including immunomodulatory drugs such as thalidomide and lenalidomide in multiple myeloma (MM), about 20-25% of patients are still considered at high risk for treatment failure. The emergence of therapeutically recalcitrant cases and disease relapse even under intensive treatment regimens suggests the existence of a dormant myeloma-initiating population within the bone marrow (BM) that is capable of drug escape. These myeloma-initiating cells are defined by the lack of the plasma cell marker CD138, while they express several hematopoietic stem (CD38) and B cell markers such as CD27 and CD19; however, the molecular and cellular mechanisms that regulate myeloma-initiating cell generation and maintenance are so far poorly understood. Recently, we and other groups showed that both NOTCH signaling and inflammation- responsive ADAR1 activation are crucial events regulating malignant stem cell maintenance in the bone marrow microenvironment, characterized by enhanced survival and self-renewal and cell cycle alterations of dormant progenitor cells. In this context, the central hypothesis of this proposal is that MM niche-derived pro- inflammatory signals induce aberrant human-specific RNA editing driven by adenosine deaminase acting on dsRNA-1 (ADAR1) in dormant myeloma-initiating cells that is accentuated by lenalidomide resistance. This project will: 1) determine whether ADAR1 activity is enhanced in myeloma-initiating cells and investigate the effects of lenalidomide treatment on ADAR1-dependent RNA editing in therapeutic resistance and relapse; 2) identify the NOTCH-regulated pro-inflammatory cytokines that activate ADAR1-dependent RNA editing in myeloma-initiating cells; and 3) determine whether direct inhibition of ADAR1 activity in MM initiating cells, or blocking microenvironmental signals that activate ADAR1, sensitizes myeloma-initiating cells to lenalidomide and prevents myeloma-initiating cell maintenance. These aims will address PQC2: What molecular or cellular events establish tumor dormancy after treatment and what leads to recurrence? We will utilize both in vitro and in vivo measures of myeloma-initiating cell function using multicolor flow cytometry and fluorescent RNA probe-based strategies to purify and profile primary MM cellular constituents, coupled with a novel diagnostic qPCR-based assay to detect endogenous RNA editing, and gene knockout (CRISPR) or lentiviral shRNA- knockdown strategies to modulate NOTCH-dependent ADAR1 activation. A fluorescent ubiquitination cell cycle indicator (FUCCI) bi-cistronic lentiviral reporer will facilitate investigations of dormant live cells, in robust bone marrow stromal co-culture models and bioluminescent humanized MM mouse models. The ultimate goal is to investigate niche-dependent ADAR1 activation as a novel mechanism driving transcriptome recoding and molecular evolution of dormant myeloma-initiating cells, laying the groundwork for targeted therapeutics with potential applications in an array of other therapeutically recalcitrant malignancies.